Functional characterization of the chlorzoxazone 6-hydroxylation activity of human cytochrome P450 2E1 allelic variants in Han Chinese

Ting Wang; Huihui Du; Jingsong Ma; Lu Shen; Muyun Wei; Xianglong Zhao; Luan Chen; Mo Li; Guorong Li; Qinghe Xing; Lin He; Shengying Qin

doi:10.7717/peerj.9628

Functional characterization of the chlorzoxazone 6-hydroxylation activity of human cytochrome P450 2E1 allelic variants in Han Chinese

PeerJ. 2020 Jul 31:8:e9628. doi: 10.7717/peerj.9628. eCollection 2020.

Authors

Ting Wang¹, Huihui Du¹, Jingsong Ma¹, Lu Shen¹, Muyun Wei¹, Xianglong Zhao¹, Luan Chen¹, Mo Li¹, Guorong Li², Qinghe Xing³, Lin He^{1

4}, Shengying Qin^{1

5}

Affiliations

¹ Bio-X Institutes, Shanghai Jiaotong University, Shanghai, China.
² School of Life Sciences, Shandong Normal University, Shandong, China.
³ Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
⁴ Baoan Maternal and Child Health Hospital, Jinan University, Guangdong, China.
⁵ Collaborative Innovation Center, Jining Medical University, Shandong, China.

Abstract

Backgrounds: Cytochrome P450 (P450) 2E1 is one of the primary enzymes responsible for the metabolism of xenobiotics, such as drugs and environmental carcinogens. The genetic polymorphisms of the CYP2E1 gene in promoter and coding regions have been identified previously in the Han Chinese population from four different geographic areas of Mainland China.

Methods: To investigate whether genetic variants identified in the CYP2E1 coding region affect enzyme function, the enzymes of four single nucleotide polymorphism (SNP) variants in the coding region (novel c.1009C>T, causing p.Arg337X, where X represents the translational stop codon; c.227G>A, causing p.Arg76His; c.517G>A, yielding p.Gly173Ser; and c.1263C>T, presenting the highest allele frequency), two novel alleles (c.[227G>A;1263C>T] and c.[517G>A;1263C>T]), and the wild-type CYP2E1 were heterologously expressed in COS-7 cells and functionally characterized in terms of expression level and chlorzoxazone 6-hydroxylation activity. The impact of the CYP2E1 variant sequence on enzyme activity was predicted with three programs: Polyphen 2, PROVEAN and SIFT.

Results: The prematurely terminated p.Arg337X variant enzyme was undetectable by western blotting and inactive toward chlorzoxazone 6-hydroxylation. The c.1263C>T and c.[517G>A;1263C>T] variant enzymes exhibited properties similar to those of the wild-type CYP2E1. The CYP2E1 variants c.227G>A and c.[227G>A;1263C>T] displayed significantly reduced enzyme activity relative to that of the wild-type enzyme (decreased by 42.8% and 32.8%, respectively; P < 0.01). The chlorzoxazone 6-hydroxylation activity of the c.517G>A transfectant was increased by 31% compared with the wild-type CYP2E1 enzyme (P < 0.01). Positive correlations were observed between the protein content and enzyme activity for CYP2E1 (P = 0.0005, r ² = 0.8833). The characterization of enzyme function allelic variants in vitro was consistent with the potentially deleterious effect of the amino acid changes as determined by prediction tools.

Conclusions: These findings indicate that the genetic polymorphisms of CYP2E1, i.e., c.1009C>T (p.Arg337X), c.227G>A (p.Arg76His), and c.517G>A (p.Gly173Ser), could influence the metabolism of CYP2E1 substrates, such as chlorzoxazone.

Keywords: Chlorzoxazone; Enzyme activity; Genetic polymorphisms; Metabolism; Cytochrome P450 2E1 (CYP2E1).

Grants and funding

This work was supported by grants from the 863 Program (No. 2012AA02A515, No. 2012AA021802), the National Nature Science Foundation of China (No. 81773818, No. 81273596, No. 30900799, and No. 81671326), the National Key Research and Development Program (No. 2017YFC0909303, No. 2016YFC0905000, No. 2016YFC0905002, No. 2016YFC1200200, and No. 2016YFC0906400), the 4th Three-year Action Plan for Public Health of Shanghai (No. 15GWZK0101), the Shanghai Pujiang Program (No. 17PJD020), and the Shanghai Key Laboratory of Psychotic Disorders (No. 13dz2260500). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.